U.S. patent application number 11/630954 was filed with the patent office on 2008-06-05 for viral particle-like construct and method of forming the same under physiological conditions.
Invention is credited to Hiroshi Handa, Masaaki Kawano, Akira Nakanishi.
Application Number | 20080131928 11/630954 |
Document ID | / |
Family ID | 35782972 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131928 |
Kind Code |
A1 |
Handa; Hiroshi ; et
al. |
June 5, 2008 |
Viral Particle-Like Construct and Method of Forming the Same Under
Physiological Conditions
Abstract
There is provided a novel method of forming uniform viral
particles under physiological conditions. The method of forming
uniform-sized viral particle aggregates composed of viral protein
is characterized by incubating a viral protein such as SV40 virus
VP1 at pH 5.0 to 7.0, room temperature, in the presence of 130 mM
to 170 mM sodium chloride and 1.5 mM to 2.5 mM divalent cation, and
in the presence of a particle formation acceleration factor such as
SV40 VP2. For encapsulation of a substance to be encapsulated in
the viral particles, the substance to be encapsulated is included
during the incubation.
Inventors: |
Handa; Hiroshi; (Kanagawa,
JP) ; Nakanishi; Akira; (Kanagawa, JP) ;
Kawano; Masaaki; (Kanagawa, JP) |
Correspondence
Address: |
Millen White Zelano Branigan PC
2200 Clarendon Boulevard, Suite 1400
Arlington
VA
22201
US
|
Family ID: |
35782972 |
Appl. No.: |
11/630954 |
Filed: |
June 30, 2005 |
PCT Filed: |
June 30, 2005 |
PCT NO: |
PCT/JP05/12524 |
371 Date: |
December 28, 2006 |
Current U.S.
Class: |
435/69.1 ;
435/235.1; 435/471 |
Current CPC
Class: |
Y02A 50/467 20180101;
A61K 2039/5258 20130101; Y02A 50/30 20180101; C12N 2710/22022
20130101; C12N 7/00 20130101; A61K 39/12 20130101; A61P 43/00
20180101; C07K 14/005 20130101; C12N 2710/22023 20130101 |
Class at
Publication: |
435/69.1 ;
435/235.1; 435/471 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C12N 7/00 20060101 C12N007/00; C12N 15/87 20060101
C12N015/87 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 1, 2004 |
JP |
2004-195822 |
Claims
1. A uniform-sized viral particle-like structure composed of a
viral protein and a particle formation acceleration factor.
2. A uniform-sized viral particle-like structure composed of a
viral protein and a particle formation acceleration factor, which
houses a substance to be encapsulated.
3. A viral particle-like structure according to claim 1, wherein
the viral protein is VP1 capsid protein of SV40 virus, JC virus or
BK virus.
4. A viral particle-like structure according to claim 3, wherein
the SV40 virus protein is VP1 capsid protein or a mutant
thereof.
5. A viral particle-like structure according to claim 4, wherein
the VP1 capsid protein mutant is a protein which is VP1 capsid
protein having the amino acid sequence listed as SEQ ID NO: 2 with
a deletion, addition or amino acid substitution of one or more
amino acids.
6. A viral particle-like structure according to claim 5, wherein
the substitution is at least one amino acid substitution from among
Glu at position 49, Glu at position 51, Glu at position 160, Glu at
position 163, Ser at position 216, Lys at position 217, Glu at
position 219, Glu at position 332, Glu at position 333 and Asp at
position 348 of the amino acid sequence listed as SEQ ID NO: 2.
7. A viral particle-like structure according to any one of claim 1,
wherein the particle formation acceleration factor is the viral
particle capsid protein, the N-terminal region of the protein
having particle formation accelerating activity, or a protein which
is modified with a deletion, addition and/or amino acid
substitution of one or more amino acids of the protein and which
retains particle formation accelerating activity.
8. A viral particle-like structure according to claim 7, wherein
the viral particle capsid protein is the capsid protein VP2 of SV40
virus, JC virus or BK virus.
9. A viral particle-like structure according to claim 7, wherein
the viral particle capsid protein is the SV40 viral capsid protein
VP2 having the amino acid sequence listed as SEQ ID NO: 1.
10. A viral particle-like structure according to claim 7, wherein
the viral particle capsid protein is a portion of the capsid
protein VP2 of SV40 virus comprising at least amino acids 1 to 272
of the amino acid sequence listed as SEQ ID NO: 1.
11. A viral particle-like structure according to claim 7, wherein
the viral particle capsid protein is a portion of the capsid
protein VP2 of SV40 virus comprising at least amino acids 1 to 58,
or 59 to 118, or 119 to 152, or 153 to 272 of the amino acid
sequence listed as SEQ ID NO: 1.
12. A viral particle-like structure according to claim 7, wherein
the viral particle capsid protein is a portion of the capsid
protein VP2 of SV40 virus comprising at least the amino acid
sequence of the VP2-binding region from residues 273 to 307 of the
amino acid sequence listed as SEQ ID NO: 1.
13. A viral particle-like structure according to claim 2, wherein
the substance to be encapsulated is a bioactive substance or
non-bioactive substance, or a mixture thereof.
14. A viral particle-like structure according to claim 2, wherein
the non-bioactive substance is a low molecular substance, a high
molecular substance or a mixture thereof.
15. A viral particle-like structure according to claim 13, wherein
the bioactive substance is a nucleic acid, protein or low molecular
substance.
16. A method of forming uniform-sized viral particle aggregates
composed of viral protein and a particle formation acceleration
factor, characterized by incubating viral protein at pH 5 to 10 at
room temperature, in the presence of 130 mM to 500 mM monovalent
cation and 2 mM to 50 mM divalent cation, and in the presence of a
particle formation acceleration factor.
17. A method of forming uniform-sized viral particle aggregates
composed of a substance to be encapsulated, and a viral protein and
a particle formation acceleration factor which surrounds it,
characterized by incubating viral protein and the substance to be
encapsulated at pH 5 to 10 at room temperature, in the presence of
130 mM to 500 mM monovalent cation and 2 mM to 50 mM divalent
cation, and in the presence of a particle formation acceleration
factor.
18. A method according to claim 16, wherein the monovalent cation
is sodium ion.
19. A method according to claim 16, wherein the divalent cation is
calcium ion.
20. A method according to claim 16, wherein the concentration of
the monovalent cation is 150 mM, and the concentration of the
divalent cation is 2 mM.
21. A method according to claim 16, wherein the viral protein is
the VP1 capsid protein of SV40 virus, JC virus or BK virus.
22. A method according to claim 16, wherein the particle formation
acceleration factor is the viral particle capsid protein, the
N-terminal region of the protein having particle formation
accelerating activity, or a protein which is modified with a
deletion, addition and/or amino acid substitution of one or more
amino acids of the protein and which retains particle formation
accelerating activity.
23. A method according to claim 22, wherein the viral particle
capsid protein is the capsid protein VP2 of SV40 virus, JC virus or
BK virus.
24. A method according to claim 22, wherein the viral particle
capsid protein is SV40 viral capsid protein VP2 having the amino
acid sequence listed as SEQ ID NO: 1.
25. A method according to claim 23, wherein the viral particle
capsid protein is a portion of the capsid protein VP2 of SV40 virus
comprising at least amino acids 1 to 272 of the amino acid sequence
listed as SEQ ID NO: 1.
26. A method according to claim 23, wherein the viral particle
capsid protein is a portion of the capsid protein VP2 of SV40 virus
comprising at least amino acids 1 to 58, or 59 to 118, or 119 to
152, or 153 to 272 of the amino acid sequence listed as SEQ ID NO:
1.
27. A method according to claim 17, wherein the substance to be
encapsulated is a bioactive substance or non-bioactive substance,
or a mixture thereof.
28. A method according to any claim 17, wherein the non-bioactive
substance is a low molecular substance, a high molecular substance
or a mixture thereof.
29. A method according to claim 27, wherein the bioactive substance
is a nucleic acid, protein or low molecular substance.
30. A method of introducing a bioactive substance into virus-like
particles composed of viral protein, characterized by coexpressing
in host cells the viral protein and capsid protein VP2 or a portion
thereof comprising the binding region of the viral protein and
having a bioactive substance linked thereto.
31. A method according to claim 30, wherein the viral protein is
SV40 virus VP1 capsid protein or a mutant thereof, and the VP2
protein is a portion of SV40 virus capsid protein VP2 comprising at
least the amino acid sequence of the VP1-binding region from
positions 273 to 307 of the amino acid sequence listed as SEQ ID
NO: 1.
32. A composition for introduction of a bioactive substance into
cells, whose active component is a viral particle structure
according to claim 13.
33. A method for producing viral particle aggregates composed of
viral protein encapsulating a polymer with a negatively charged
surface, the method being characterized by mixing the viral protein
with the polymer at 0.01 to 100 parts (by weight) with respect to
360 parts of the viral capsid protein, and dialyzing the mixture
against an aqueous solution containing a monovalent metal salt and
a divalent metal salt.
34. A method according to claim 33, wherein the negatively charged
polymer is DNA, RNA or a synthetic nucleic acid-like structure.
35. A method according to claim 33, wherein in the method for
producing the viral particle aggregates, the weight ratio of the
viral protein and the negatively charged polymer added to 360 parts
of the viral capsid protein is 0.2 or greater.
36. A method according to claim 33, wherein the viral protein is
the VP1 capsid protein of SV40 virus, JC virus or BK virus.
37. A method according to claim 36, wherein the viral protein is
the SV40 virus VP1 capsid protein represented by SEQ ID NO: 2, or a
mutant thereof.
38. A method according to claim 33, wherein the monovalent metal
ion is sodium ion, and the divalent metal ion is calcium ion.
Description
TECHNICAL FIELD
[0001] The present invention relates to a viral particle-like
structure comprising viral protein, and to a method of forming it.
The viral particle-like structure can encapsulate other substances
within it, and therefore has potential use as a carrier for drug
delivery and gene therapy.
BACKGROUND ART
[0002] Conventional formation of viral particle-like structures has
involved recovering virus-like-particles formed within cells.
[0003] In some methods, the virus-like particles are purified from
the cells and are first dissociated into particle structure units
(for example, VP1 pentamers in the case of SV40 virus), and then
reconstituted into virus-like particles in a test tube.
[0004] Conventional reconstituting methods have been conducted
under non-physiological conditions with high salt concentration,
but because of problems such as inactivation or poor solvent
solubility of bioactive substances included in the particles, the
conditions have not been suitable for taking up bioactive
substances into particles. Moreover, it has been difficult to
efficiently reconstitute virus-like particles of uniform size by
such methods.
DISCLOSURE OF THE INVENTION
[0005] The present invention provides a method which allows a viral
particle-like structure to be reconstituted in a test tube to form
uniform-sized particles efficiently and under physiological
conditions.
[0006] The invention further provides a method for forming a viral
particle-like structure in host cells.
[0007] The object of the invention, of forming uniform-sized
virus-like particles efficiently under physiological conditions in
a test tube, is achieved by adding to the reconstituting
environment a protein that is found in natural viral particles.
[0008] Thus, the invention provides a uniform-sized viral
particle-like structure composed of a viral protein and a particle
formation acceleration factor. The invention further provides a
uniform-sized viral particle-like structure composed of a viral
protein and a particle formation acceleration factor, which houses
a substance to be encapsulated.
[0009] The invention still further provides a method of forming a
uniform-sized particle aggregate composed of viral protein and a
particle formation acceleration factor, characterized by incubating
the viral protein at pH 5 to 10.0, room temperature, in the
presence of 130 mM to 500 mM monovalent cation and 2 .mu.M to 50 mM
divalent cation, and in the presence of the particle formation
acceleration factor; and a method of forming a uniform-sized viral
particle-like structure composed of a substance to be encapsulated
and a viral protein surrounding it, with a particle formation
acceleration factor, characterized by incubating the viral protein
and substance to be encapsulated at pH 5 to 10.0, room temperature,
in the presence of 130 mM to 500 mM monovalent cation and 2 .mu.M
to 50 mM divalent cation, and in the presence of the particle
formation acceleration factor.
[0010] The invention still further provides a method of introducing
a bioactive substance into virus-like particles composed of viral
protein and capsid protein VP2 or a portion thereof, characterized
by coexpressing in host cells the viral protein and capsid protein
VP2 or a portion thereof comprising the binding region of the viral
protein and having a bioactive substance linked thereto.
[0011] The invention still further provides a method for producing
viral particle aggregates composed of viral protein encapsulating a
polymer with a negatively charged surface, the method being
characterized by mixing the viral protein with the polymer at 0.01
to 100 parts (by weight) with respect to 360 parts of the viral
capsid protein, and dialyzing the mixture against an aqueous
solution containing a monovalent metal salt and a divalent metal
salt.
[0012] The negatively charged polymer is preferably DNA, RNA or a
synthetic nucleic acid-like structure. In the method for producing
viral particle aggregates, the weight ratio of the viral protein
and the negatively charged polymer added to 360 parts of the viral
capsid protein is preferably 0.2 part or greater.
[0013] The viral protein of the viral particle-like structure is
preferably VP1 capsid protein of SV40 virus, JC virus or BK
virus.
[0014] As SV40 viral proteins there may be mentioned VP1 capsid
protein and its mutant forms. Examples of VP1 capsid protein
mutants include a protein which is VP1 capsid protein having the
amino acid sequence listed as SEQ ID NO: 2 with a deletion,
addition or amino acid substitution of one or more amino acids.
Examples of specific substitutions include at least one amino acid
substitution from among Glu at position 49, Glu at position 51, Glu
at position 160, Glu at position 163, Ser at position 216, Lys at
position 217, Glu at position 219, Glu at position 332, Glu at
position 333 and Asp at position 348 of the amino acid sequence
listed as SEQ ID NO: 2.
[0015] The particle formation acceleration factor of the viral
particle-like structure is preferably the viral particle capsid
protein, the N-terminal region of the protein having particle
formation accelerating activity, or a protein which is modified
with a deletion, addition and/or amino acid substitution of one or
more amino acids of the protein and which retains particle
formation accelerating activity. The viral particle capsid protein
is preferably the capsid protein VP2 of SV40 virus, JC virus or BK
virus. As a more specific example, the viral particle capsid
protein may be capsid protein VP2 of SV40 virus having the amino
acid sequence listed as SEQ ID NO: 1.
[0016] When the virus-like structure of the invention is formed in
vitro, such as in a test tube, preferably the viral particle capsid
protein is a portion of SV40 viral capsid protein VP2 comprising at
least the amino acid sequence from residues 1 to 272, or at least
the amino acid sequence from residues 1 to 58, 59 to 118, 119 to
152 or 153 to 272 of the amino acid sequence listed as SEQ ID NO:
1.
[0017] When the virus-like structure of the invention is formed in
host cells, preferably the viral particle capsid protein is a
portion of SV40 viral capsid protein VP2 comprising at least the
amino acid sequence of the VP1-binding region from residues 273 to
307 of the amino acid sequence listed as SEQ ID NO: 1, and
preferably it is linked to the desired bioactive substance or
non-bioactive substance to be introduced, or a combination
thereof.
[0018] The non-bioactive substance is, for example, a low molecular
substance or high-molecular substance, or a combination
thereof.
[0019] The factor to be encapsulated will typically be a bioactive
substance, and for example, may be a nucleic acid, protein or low
molecular substance.
[0020] The monovalent cation forming the viral particle-like
structure of the invention is preferably sodium ion, and it may be
used in the form of sodium chloride. The divalent cation forming
the viral particle-like structure of the invention is preferably
calcium ion, and it may be used in the form of calcium chloride.
The concentration of the monovalent cation may be, for example, 150
mM, and the concentration of the divalent cation may be, for
example, 2 mM.
[0021] The invention also relates to a composition for introduction
of a bioactive substance into cells, whose active component is the
aforementioned viral structure comprising a bioactive molecule.
BRIEF EXPLANATION OF THE DRAWINGS
[0022] FIG. 1 is a set of electron microscope photographs showing a
viral particle-like structure formed by the method of the invention
in the presence of a particle formation acceleration factor.
[0023] FIG. 2 is a set of electron microscope photographs showing
viral protein treated under physiological conditions in the absence
of a particle formation acceleration factor.
[0024] FIG. 3 is a set of electron microscope photographs showing
formation of viral particle-like structures upon changing the
proportion of viral protein VP1 and particle formation acceleration
factor VP2.
[0025] FIG. 4 is a pair of electron microscope photographs showing
viral particle-like structures produced using particle formation
acceleration factor VP2 lacking the C-terminal end.
[0026] FIG. 5 is a set of electron microscope photographs showing
viral particle-like structures produced using particle formation
acceleration factor VP2 having a point mutation introduced at the
C-terminal end.
[0027] FIG. 6 is a set of electron microscope photographs showing
that uniform globular viral particle-like structures are formed
even in the absence of a particle formation acceleration factor
when incubation is performed under non-physiological conditions of
pH 8 to 10.
[0028] FIG. 7 is an image showing the results of fractionation by
sucrose density centrifugal separation and detection by Southern
blotting of the viral particles prepared in Example 2.
[0029] FIG. 8 is an image showing distribution of pEG DNA after
fractionation of virus-like particles composed of VP1-VP2 protein
in the presence of pEG by sucrose density gradient centrifugation
in Example 3, and distribution of protein detected with anti-VP1
antibody.
[0030] FIG. 9 is a photograph showing that pEG introduced into
COS-1 cells by virus-like particles in Example 3 were expressed in
cells and that fluorescent protein was produced.
[0031] FIG. 10 is a diagram showing the results of Example 4, by
the relationship between portions of VP2 protein as a particle
formation acceleration factor, and virus formation.
[0032] FIG. 11 is a diagram showing the results of Example 5 which
indicate the portion of VP2 necessary for taking up into VP1
virus-like structures.
[0033] FIG. 12 is a set of line graphs showing the results of
Example 6, indicating that DNA is taken up into virus-like
structures formed from VP1 under prescribed conditions.
[0034] FIG. 13 is a set of photographs showing the results of
electron microscope observation of the product obtained in Example
6.
[0035] FIG. 14 is photograph showing reconstitution by mixing RNase
pre-treated total RNA, RNase untreated total RNA and purified VP1
protein and dialyzing the mixture in a pH 5, 150 mM NaCl, 2 mM
CaCl.sub.2 solution for 16 hours at room temperature, in Example 7.
After reconstitution, an electron microscope was used for
observation of the aggregated VP1 pentamers in exchanged solvent.
This photograph shows the RNase-treated RNA as RNase+ and the
RNase-untreated RNA as RNase-. It suggests that formation of
globular virus-like particles occurs due to the presence of
RNA.
BEST MODE FOR CARRYING OUT THE INVENTION
[0036] According to the invention, viruses that may be used as
protein sources for formation of viral protein particles are not
particularly restricted so long as particles can be formed from the
major constituent protein or particle outer shell constituent
protein. As examples of major constituent proteins there may be
mentioned those of Simian virus 40 (SV40), human polyoma virus JC,
and BK virus. SV40 VP1 is particularly preferred. As an example of
a particle outer shell forming protein there may be mentioned the
capsid protein VP1 (of SV40 or the human polyoma viruses JC virus
and BK virus) or the like.
[0037] The SV40 VP1 may be naturally occurring VP1 or a mutant
thereof. Examples of mutants include VP1 capsid protein having the
amino acid sequence listed as SEQ ID NO: 2 with a deletion,
addition or amino acid substitution of one or more amino acids, and
specific substitutions include at least one amino acid substitution
from among Glu at position 49, Glu at position 51, Glu at position
160, Glu at position 163, Ser at position 216, Lys at position 217,
Glu at position 219, Glu at position 332, Glu at position 333 and
Asp at position 348 of the amino acid sequence listed as SEQ ID NO:
2.
[0038] According to one mode, the invention provides a protein
(mutant A; mtA) wherein Glu at position 160 is replaced by another
amino acid, and which can form more rigid or stable virus-like
protein particles than the wild type. This Glu is preferably
replaced by Gln.
[0039] According to another mode, the invention provides a protein
(mutant B; mtB) wherein Glu at position 163 is replaced by another
amino acid, and which forms more rigid or stable virus-like protein
particles than the wild type. This Glu is preferably replaced by
Gln.
[0040] According to yet another mode, the invention provides a
protein (mutant C; mtC) wherein Asp at position 348 is replaced by
another amino acid, and which forms more rigid or stable virus-like
protein particles than the wild type. This Asp is preferably
replaced by Asn.
[0041] According to yet another mode, the invention provides a
protein (mutant D; mtD) wherein Glu at position 160 and Glu at
position 163 are replaced by other amino acids, and which forms
more rigid virus-like protein particles than the wild type. This
Glu is preferably replaced by Gln.
[0042] According to yet another mode, the invention provides a
protein (mutant E; mtE) wherein Glu at position 160, Glu at
position 163 and Asp at position 348 are replaced by other amino
acids, and which forms more rigid or stable virus-like protein
particles than the wild type. The Glu is preferably replaced by
Gln, and Asp is preferably replaced by Asn.
[0043] According to yet another mode, the invention provides a
protein (mutant F; mtF) wherein Glu at position 332, Glu at
position 333 and Asp at position 348 are replaced by other amino
acids, and which forms rod-shaped virus-like protein particles at
high incidence. The Glu is preferably replaced by Gln, and Asp is
preferably replaced by Asn.
[0044] According to yet another mode, the invention provides a
protein (mutant G; mtG) wherein Glu at position 49 and Glu at
position 51 are replaced by other amino acids, and which forms more
rigid or stable virus-like protein particles than the wild type.
The Glu is preferably replaced by Gln.
[0045] According to yet another mode, the invention provides a
protein (mutant H; mtH) wherein Glu at position 49, Glu at position
51, Glu at position 160, Glu at position 163, Ser at position 216,
Lys at position 217, Glu at position 219, Glu at position 332, Glu
at position 333 and Asp at position 348 are replaced by other amino
acids, and which does not form virus-like protein particles as
easily as the wild type. The Glu is preferably replaced by Gln, the
Asp is preferably replaced by Asn, the Ser is preferably replaced
by Ala and the Lys is preferably replaced by Ala.
[0046] A method of preparing such mutants is described in detail in
Japanese Unexamined Patent Publication No. 2002-360266.
[0047] According to the invention, it is necessary to use the
particle formation acceleration factor under conditions of pH 5 to
10. The particle formation acceleration factor is preferably, for
example, a viral particle protein. As examples of viral particle
proteins there may be mentioned capsid protein VP2 of SV40 virus,
JC virus or BK virus, or its N-terminal portion, or histone protein
or the like. Particularly preferred as a viral particle protein is
SV40 VP2 or its N-terminal portion. The amino acid sequence of SV40
VP2 is listed as SEQ ID NO: 1. When SV40 VP2 or a portion thereof
is used as the particle formation acceleration factor for the
invention to form a virus-like structure in vitro, it need only
comprise at least the amino acid sequence from the amino acid at
position 1 to the amino acid at position 58, the amino acid
sequence from the amino acid at position 59 to position 118, the
amino acid sequence from positions 119 to 152 and the amino acid
sequence from positions 153 to 272, of the amino acid sequence
listed as SEQ ID NO: 1.
[0048] When SV40 virus capsid protein VP2 is used to form the
virus-like structure in cells, the capsid protein need only
comprise at least the amino acid sequence of the VP1-binding region
from positions 273 to 307 of the amino acid sequence listed as SEQ
ID NO: 1.
[0049] The viral particle protein of the invention may have, for
example, the amino acid sequence listed as SEQ ID NO: 1 or its
N-terminal sequence modified by an addition, deletion and/or amino
acid substitution of one or more amino acid residues, while still
retaining particle formation acceleration factor activity. The
number of amino acid residues modified may be, for example, 1 to
20, 1 to 15 or one to a few.
[0050] The concentration of the viral protein forming the outer
shell of the particles is 50 ng/.mu.L to 500 ng/.mu.L and
preferably 70 ng to 200 ng, and the concentration of protein as the
particle formation acceleration factor is 1 ng/.mu.L to 1
.mu.g/.mu.L and preferably 10 ng/.mu.L to 100 ng/.mu.L. The
concentration of the substance to be encapsulated, for
encapsulation into the viral particles, will differ depending on
the type of substance but may be 0.1 ng/.mu.L to 10 .mu.g/.mu.L and
preferably 10 ng/.mu.L to 1 .mu.g/.mu.L.
[0051] According to the invention, the viral protein may be
incubated (1) in a pH range of pH 5 to 10, (2) at room temperature
and in the presence of (3) 130 mM to 500 mM monovalent cation, (4)
2 .mu.M to 50 mM divalent cation and (5) a particle formation
acceleration factor, to form globular, uniform-sized particles.
Sodium is preferred as the monovalent cation, such as in the form
of sodium chloride, and the concentration of sodium ion is
preferably 140 mM to 160 mM and especially 150 mM.
[0052] As divalent cations there may be used calcium ion, cadmium
ion, manganese ion, magnesium ion and zinc ion, but calcium ion is
particularly preferred, and for example, calcium chloride may be
used. The concentration of calcium ion is preferably 1.75 mM to
2.25 mM, and especially 2 mM.
[0053] Incubation in a range of pH 8 to 10 at room temperature in
the presence of 130 mM to 170 mM sodium chloride, 1.5 mM to 2.5 mM
divalent cation can form globular uniform-sized viral particle-like
structures without addition of a particle formation acceleration
factor.
[0054] In the method for formation of viral particles encapsulating
a substance to be encapsulated according to the invention, the
substance to be encapsulated may be included during the incubation
for formation of the viral particles. There are no particular
restrictions on the substance to be encapsulated, and for example,
there may be mentioned nucleic acid, i.e. DNA or RNA, and
especially DNA, proteins or peptides, and various low molecular
substances such as pharmaceutically active substances.
[0055] The viral structure comprising the bioactive molecule
prepared in the manner described above may be used for introduction
of the bioactive substance into cells. This will allow application
for introduction of the bioactive substance into viable cells for
the purpose of drug delivery, gene therapy or the like, in the
field of regenerative therapies employing gene transfer, gene
therapy, targeted gene expression and functional suppression, or
application for tissue- and organ-specific or lesion-specific
labeling methods using virus-like particles containing labeled
substances and the like.
EXAMPLES
[0056] The present invention will now be explained in greater
detail by examples.
Example 1
Preparation of Viral Particles
(1) Preparation of Viral Particle (VP1) Pentamers
[0057] After seeding Sf9 cells at 1.times.10.sup.7 each into fifty
10 cm-diameter tissue culturing dishes, they were infected with
recombinant baculovirus expressing SV40 viral protein (VP1) with a
m.o.i. (multiplicity of infection) of 5 to 10.
[0058] At 72 hours after infection, the cells were recovered with
medium using a scraper, and rinsed twice with cooled phosphate
buffered saline (PBS). To the recovered cells there was added 10 mL
of ice-cooled sonication buffer (20 mM Tris-HCl (pH 7.9), 1% (w/v)
sodium deoxycholate (DOC), 2 mM PMSF), and then a VP-15S
(sonicator) by Taitec was used for 10 minutes of ultrasonic
disruption while cooling on ice under conditions with a 50% duty
cycle and output at 5. The cell disruptate was centrifuged at
14,000 g, 4.degree. C. for 20 minutes and the supernatant was
recovered.
[0059] Cesium chloride solutions with four different densities
(50%, 40%, 30%, 20% (w/v)) were gently layered at 1.5 ml each in a
SW41Ti Open Top Ultraclear Tube (Beckman) in order from the highest
density, and then 5 mL of the cell disruptate was layered thereover
and centrifugation was performed for 2.5 hours with a SW41Ti Rotor
(Beckman) at 30,000 rpm, 4.degree. C. After centrifugation, the
white SV40 virus-like particle (VLP) layer formed midway in the
density gradient was collected. The collected solution was
transferred to an SW55Ti Open Top Ultraclear Tube (Beckman), a 37%
(w/v) cesium chloride solution was added to approximately 5 mm from
the tip of the volume tube, and the mixture was centrifuged for 20
hours with an SW55Ti Rotor (Beckman) at 50,000 rpm, 4.degree. C.
after which the re-formed VLP layer was recovered.
[0060] To the obtained purified viral protein solution there was
added a 1/100 volume of 10% (v/v) surfactant NP-40 (final
concentration: 0.1%), and the mixture was dialyzed for 24 hours at
4.degree. C. in a dialyzing solution containing 20 mM Tris-HCl (pH
7.9), 0.1% NP-40 for removal of the cesium chloride. Dialysis was
followed by centrifugation for 10 minutes at 15,000 g, 4.degree.
C., and the supernatant was collected.
[0061] Next, 0.25 M ethylene glycol bis (.beta.-aminoethyl
ether)-N,N,N',N'-tetraacetate (EGTA) and 1 M dithiothreitol (DTT)
were added to the virus-like particles to respective final
concentrations of 25 mM EGTA and 30 mM DTT, and incubation was
performed at 37.degree. C. for 1 hour to dissociate the virus-like
particles into VP1 pentamers. Incubation was followed by
centrifugation for 10 minutes at 15,000 g, 4.degree. C., and the
obtained supernatant was subjected to gel filtration chromatography
for purification of the VP1 pentamers. The chromatography was
carried out using a HiLoad 16/60 Superdex 200 pg column (Pharmacia)
under conditions of 20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 5 mM
EGTA, 5 mM DTT, 4.degree. C.
[0062] A portion of each obtained fraction was taken and subjected
to SDS polyacrylamide gel electrophoresis, protein detected at a
molecular weight of about 200 kDa was considered to be VP1
pentamer, and the fraction containing the protein was considered to
be the VP1 pentamer-containing fraction and was frozen with liquid
nitrogen and then stored at -80.degree. C.
(2) Preparation of SV40-VP2 Protein
[0063] The SV40-VP2 gene having the histidine sequence and FLAG
sequence inserted at the amino terminal end was incorporated into
pET-14b vector for transformation of E. coli BL21. The transformed
E. coli was inoculated into 250 ml of LB medium and shake cultured
at 37.degree. C. When the culture solution reached the logarithmic
growth stage (turbidity: O.D. value=0.3 (wavelength: 660 nm)),
protein expression was induced with IPTG. At four hours after
induction of expression, the E. coli cells were centrifuged and
collected and then rinsed twice with cooled phosphate buffered
saline (PBS). To the recovered E. coli cells there was added 40 ml
of ice-cooled binding buffer (20 mM Tris-HCl (pH 7.9), 10%
glycerol, 500 mM KCl, 0.2 mM EDTA, 0.1% NP-40, 0.5 mM DTT, 10 mM
imidazole), and a VP-15S (sonicator) by Taitec was used for 10
minutes of ultrasonic disruption while cooling on ice under
conditions with a 50% duty cycle and output at 5. The cell
disruptate was then centrifuged for 20 minutes at 14,000 g,
4.degree. C. and the supernatant was collected.
[0064] The collected supernatant was mixed with 500 .mu.L of His
resin (Qiagen) that had been equilibrated with binding buffer, and
the mixture was stirred by slowly rotating with a rotor for one
hour at 4.degree. C. The stirred solution was centrifuged to
convert the resin to a pellet, and the supernatant was removed.
After adding 10 mL of wash buffer (20 mM Tris-HCl (pH 7.9), 10%
glycerol, 500 mM KCl, 0.2 mM EDTA, 0.1% NP-40, 0.5 mM DTT, 20 mM
imidazole) to the resin, the mixture was stirred. The stirred
solution was again centrifuged and the supernatant was carefully
removed. This washing procedure was repeated 3 times. Finally, 500
.mu.L of elution buffer (20 mM Tris-HCl (pH 7.9), 10% glycerol, 500
mM KCl, 0.2 mM EDTA, 0.1% NP-40, 0.5 mM DTT, 1M imidazole) was
added to the resin and the mixture was stirred. The stirred
solution was centrifuged and the supernatant was carefully
collected. This procedure was repeated twice to obtain a total of 1
ml of SV40-VP2 protein.
(3) In Vitro Reconstitution of Viral Particles Under Physiological
Conditions
[0065] The prepared SV40-VP1 protein pentamers and SV40-VP2 protein
were used for in vitro reconstitution of viral particles under
physiological conditions. Specifically, for pH 5 to 7, 3.4 .mu.l of
800 ng/.mu.L VP2 protein was added to 150 .mu.L of 82.5 ng/.mu.L
VP1 pentamer protein, incubation was performed at 4.degree. C. for
30 minutes, and the mixture was dialyzed by a dialysis method with
a solution containing 150 mM NaCl, 2 mM CaCl.sub.2 for
reconstitution. The addition was to a molar ratio of VP1 protein
and VP2 protein of 360:84. Detection of viral-like particles was
accomplished by electron microscope observation. The results are
shown in FIG. 1.
(4) Examination of VP1 Pentamer Assembly Under Physiological
Conditions
[0066] A solution containing the purified VP1 pentamer protein was
solvent-exchanged under physiological conditions, and the state of
aggregation was examined. Unlike the results of (3) in which
particle formation was observed, in this case when the pH was 5.0
to 7.0 under physiological conditions, almost no viral
particle-like structure formation was seen with VP1 pentamer alone.
VP1, for example, 150 .mu.L of VP1 pentamer at 270 ng/.mu.L
concentration, was dialyzed at room temperature with a solution of
150 mM NaCl, 2 mM CaCl.sub.2 at pH 4, 5, 6 or 7. After 16 hours
from the start of dialysis, the solution was recovered and observed
under an electron microscope, and the state of aggregation of VP1
pentamers under different conditions was observed. The results are
shown in FIG. 2.
(5) Aggregation with Addition of VP2 Protein to VP1 Pentamer at
Varying Proportions Under Physiological Conditions
[0067] Protein solution was added to purified VP1 pentamer protein
at molecular weight ratios of VP1 protein:VP2 protein=360:10.5,
360:21, 360:42 and 360:84. The solution was solvent-exchanged under
physiological conditions at pH 5.0 at room temperature using a
dialysis method. The exchanged solvent was observed under an
electron microscope to examine the state of aggregation seen when
varying the VP2 protein concentration.
[0068] For example, protein solutions were mixed with 150 .mu.L of
VP1 pentamer at 270 ng/.mu.L concentration and 1.1 .mu.L, 2.2
.mu.L, 4.4 .mu.L and 8.8 .mu.L of VP2 protein at 1.1 .mu.g/.mu.L
concentration. The solution was incubated at 4.degree. C., 30 min
and dialyzed for 16 hours at room temperature with a pH 5, 150 mM
NaCl, 2 mM CaCl.sub.2 solution. The solution was recovered and
observed under an electron microscope to observe the state of
aggregation of VP1 pentamers with varying VP2 protein
concentration. The results are shown in FIG. 3. Characteristic
rod-shaped structures were seen under these pH conditions in
several of the experiment groups, but globular virus-like particles
were simultaneously formed.
(6) Aggregation of VP1 Pentamers with Addition of .DELTA.C13 VP2
Protein, AC40 VP2 Protein and AC80 VP2 Protein Lacking Carboxyl
Terminal Ends
[0069] Purified VP1 pentamer protein and purified .DELTA.C40 VP2
protein (molecular weight: approximately 34 kDa) and AC80 VP2
protein (molecular weight: approximately 30 kDa) were combined in a
molecular weight ratio of PV1:carboxyl terminal-lacking VP2=360:84,
and the mixture was incubated at 4.degree. C. for 30 minutes. The
mixture was dialyzed at room temperature with a pH 5.0, 150 mM
NaCl, 2 mM CaCl.sub.2 solution.
[0070] For example, 2.6 .mu.L of AC40 VP2 at 763 ng/.mu.L
concentration or 2.3 .mu.L of AC80VP2 at 758 ng/.mu.L concentration
was added to 150 .mu.L of VP1 at 75.7 ng/.mu.L concentration, and
the mixture was incubated for 30 minutes at 4.degree. C. The
mixture was dialyzed for 16 hours at room temperature using the
aforementioned solution, and then the mixture was recovered and
observed under an electron microscope to examine the state of
aggregation of VP1 pentamers with addition of carboxyl
terminal-lacking VP2 protein. The results are shown in FIG. 4. It
is seen that virus-like particle formation can be induced despite
the absence of a portion of the VP2 amino acid sequence.
(7) Observation of VP1 pentamer assembly with addition of point
mutation-introduced VP2 protein VP1 pentamer protein was mixed with
VP2 protein having different point mutations, specifically, VP2
protein having the Pro, Gly, Gly from positions 283 to 285 mutated
to Arg, Glu, Arg (hereinafter, PGP.fwdarw.RER), the Phe at position
276 and Ile at position 277 mutated to Glu (hereinafter,
FI.fwdarw.EE), or to Ala (hereinafter, FI.fwdarw.AA), and the Leu
at position 296 and Leu at position 300 mutated to Ala
(hereinafter, LPLLL.fwdarw.APLLA), in a molecular weight ratio of
VP1:point mutated VP2=360:84, and each mixture was allowed to stand
at 4.degree. C. for 30 minutes and then dialyzed against a pH 5.0,
150 mM NaCl, 2 mM CaCl.sub.2 solution at room temperature.
[0071] For example, 2.7 .mu.L of PGP.fwdarw.RER VP2 at 984 ng/.mu.L
concentration, or 2.3 .mu.L of FI.fwdarw.EE VP2 at 1.18 pg/.mu.L
concentration or 3.5 .mu.L of LRLLL.fwdarw.ARLLA VP2 at 779
ng/.mu.L concentration or 2.4 .mu.L of FI.fwdarw.AA VP2 at 1.13
.mu.g/.mu.L concentration was added to 150 .mu.L of VP1 at 82.5
ng/.mu.L concentration, and the mixture was allowed to stand for 30
minutes at 4.degree. C. and dialyzed under the conditions described
above. At 16 hours after the start of dialysis, the mixture was
recovered and observed under an electron microscope, and the state
of VP1 pentamer assembly with addition of point mutation-introduced
VP2 protein was confirmed. The results are shown in FIG. 5. In all
cases, there was no inhibition against the effect of virus-like
particle formation by introduction of point mutations into VP2.
(8) Observation of VP1 Pentamer Assembly Under Conditions of pH 8.0
to pH 10.0.
[0072] A solution containing purified VP1 pentamer protein was
dialyzed under conditions of pH 8.0 to pH 10.0 and the state of
aggregation was examined. Virus-like particle formation was
observed even with VP1 pentamer alone at pH 8.0 to 10.0 under
physiological conditions, unlike with the conditions of pH 5.0 to
pH 7.0 necessary for particle formation acceleration factor. For
example, 150 .mu.L of VP1 pentamer at 270 ng/.mu.L concentration
was dialyzed with 150 mM NaCl, 2 mM CaCl.sub.2 solutions at pH 8, 9
or 10 at room temperature. After 16 hours from the start of
dialysis, the solution was recovered and observed under an electron
microscope, and the state of aggregation of VP1 pentamers under
different conditions was observed. The results are shown in FIG.
7.
Example 2
Formation of Virus-Like Particles Incorporating DNA
[0073] Example 1 was repeated. However, in step (3) for in vitro
reconstitution of the virus-like particles under physiological
conditions, a 3000 bp plasmid was included and the formed
virus-like particles comprising DNA incorporated into the
virus-like particles were subjected to sucrose density gradient
centrifugation, and then fractionation and Southern blotting for
detection of DNA. As shown in FIG. 7, the DNA was incorporated into
the virus-like particles.
[0074] The prepared SV40-VP1 protein pentamers and SV40-VP2 protein
were used for in vitro reconstitution of the virus-like particles
under physiological conditions. DNA was added during the procedure.
Specifically, at pH 5.0 to 7.0, for example, 2.8 .mu.L of 800
ng/.mu.L VP2 protein was added to 150 .mu.L of 75.7 ng/.mu.L VP1
pentamer protein, and then 21 .mu.L of 5.7 ng/.mu.l 3000 bp
circular double-stranded plasmid DNA (pG5vector) was added. The
mixture was incubated at 4.degree. C. for 30 minutes and dialyzed
using a dialysis method with a 150 mM NaCl, 2 mM CaCl.sub.2
solution for reconstitution.
[0075] Sucrose density gradient centrifugation was carried out in
order to confirm detection of the DNA added to the reconstituted
virus-like particle fraction. The centrifuged sample was
fractionated and the fractions were subjected to protease treatment
for decomposition of the VP1 protein. The sample was separated by
agarose electrophoresis and subjected to Southern blotting to
confirm that the DNA could be detected in the virus-like particle
fraction. The virus-like particles are usually included in
fractions #8, 9 and 10, and as shown in FIG. 7, detection of DNA in
fractions #8, 9 and 10 confirmed that DNA had been enveloped in the
virus-like particles.
Example 3
Gene Transfer into Cells Using DNA-Incorporating Virus-Like
Particles
[0076] Example 2 was repeated. However, the plasmid used was pEG
which can express a fluorescent protein (GFP) in mammalian
eukaryotic cells. Formation of virus-like particles containing the
pEG plasmid DNA was accomplished by sucrose density gradient
centrifugation, and it was confirmed that DNA was contained in the
fractions containing the virus-like particles. Specifically, the
virus-like particles were reconstituted in a solvent containing 150
mM sodium chloride, 2 mM calcium chloride and 20 mM Tris HCl (pH
7.2) using VP1 and VP2 protein in the presence of the pEG plasmid,
and the plasmid DNA-containing virus-like particles were
fractionated by sucrose density gradient centrifugation.
[0077] The virus-like particles were detected by Western blotting
using anti-VP1 antibody (a-VP1), and pEG was detected by Southern
blotting. The results are shown in FIG. 8. The numbers in the image
represent the fraction numbers, with the top density gradient
listed first and the bottom listed last, and P represents the
pellet that precipitated at the tube bottom during centrifugation.
The fact that this DNA was resistant to treatment by the DNA lyase
DNaseI suggested that it was included within the outer shell of the
VP1-VP2 protein.
[0078] These virus-like particles were used for introduction of pEG
DNA into COS-1 cells. Specifically, 6.65.times.10.sup.4 COS-1 cells
were spread on a 6 cm-diameter dish and cultured overnight. The
culture solution was removed without detaching the cells, and
approximately 100 .mu.L of virus-like particles containing the
aforementioned plasmid DNA were added to the cells. After
incubation at 37.degree. C. for 2 hours, the cells were wetted with
culture solution every 15 minutes to avoid drying of the cells.
After culturing, 1.5 mL of culture solution was added to the cells
and culturing was conducted at 37.degree. C. for 48 hours.
Expression of GFP in the cells was then confirmed with a
fluorescent microscope to confirm transfer of the plasmid DNA into
the cells. The results are shown in FIG. 9. Expression of
fluorescent protein encoded by pEG was observed in most of the
cells (The indefinite shaped white sections in FIG. 9 are cells
expressing fluorescent protein.), thus confirming a high rate of
gene transfer by the DNA-containing viral particles.
Example 4
Identification of VP2 Portion as Particle Formation Acceleration
Factor Contributing to Extracellular Formation of Virus-Like
Structure of VP1 Protein
[0079] In order to determine the region of the SV40 capsid protein
VP2 acting as a particle formation acceleration factor necessary
for formation of virus-like structures from SV40 VP1 protein, 0.44
.mu.M of the different regions of VP2 protein (full-length amino
acid sequence as listed in SEQ ID NO: 1) shown in FIG. 10 and 2.2
.mu.M of SV40 VP1 protein were incubated in a solution (pH 5.0)
containing 150 mM NaCl and 2 mM CaCl.sub.2, and the products were
observed under an electron microscope. The results are shown in
FIG. 10. In this diagram, "V" indicates that a uniform pentameric
virus-like structure had been formed, "Ti" indicates
microparticles, "(-)" indicates that no particles were formed, and
"Tu" indicates that tube-like structures were formed.
[0080] As clearly seen from the results in FIG. 10, in order for
SV40 capsid protein VP2 to function as a particle formation
acceleration factor, it must include at least the amino acid
sequence from residues 1 to 58 and the amino acid sequence from
residues 119 to 272 of the amino acid sequence of SEQ ID NO: 1.
Example 5
Determination of Region of VP2 Necessary for Intracellular
Incorporation of VP2 into Virus-Like Structures
[0081] It has been reported that co-expressing SV40 VP2 or VP3
protein (VP3 has the same C-terminal sequence as VP2) with VP1 in
insect cells results in inclusion of VP2 and VP3 inside the formed
VLP. This phenomenon was utilized to examine the possibility of
encapsulating a bioactive substance into virus-like structures
composed of VP1, by fusing GFP to VP2, VP3 or different fragments
thereof and co-expressing the fusion proteins with VP1 to examine
whether the fusion proteins were included in the virus-like
structures.
Experiment Method
[0082] The fusion proteins used were VP2 protein, VP3 protein
(partial VP2 protein) and four different C-terminal fragments of
VP2 protein (VP3 protein) comprising the VP1-binding region
(residues 273 to 307 of VP2) (total of 6 different proteins), each
fused with the GFP at the N-terminal or C-terminal end (total of 12
different fusion proteins). The structures of these 12 different
fusion proteins are illustrated in FIG. 11. Baculovirus expressing
each of these fusion proteins was prepared, and insect cells were
coinfected with each fusion protein-expressing baculovirus and
VP1-expressing baculovirus.
[0083] After 84 hours, the cells were recovered with a scraper and
rinsed with ice-cooled PBS(-). The cells were disrupted by
ultrasonic disruption. Next, 500 .mu.l of sonication buffer (20 mM
Tris-HCl (pH 7.9), 1% sodium deoxycholate (DOC), 2 mM
phenylmethylsulfonyl fluoride (PMSF), 1 .mu.g/ml chymostatin,
aprotinin, leupeptin, antipain, pepstatin) was added to each 10
cm-diameter dish, and an ultrasonic disruptor was used while
cooling on ice until the solution became transparent. After the
ultrasonic disruption, the mixture was centrifuged for 10 minutes
at 15,000.times.g, 4.degree. C. and the supernatant was used as the
cell lysate. A portion of the cell lysate was separated by
SDS-PAGE, Western blotting was performed with anti-VP1 polyclonal
antibody and anti-GFP monoclonal antibody (Roche), and expression
of VP1 protein and GFP fusion protein was confirmed.
[0084] The prepared cell lysate was combined with 20 mM Tris-HCl
(pH 7.9) to 20 .mu.l, and layered onto 0.6 ml of 20% to 40% (w/v)
sucrose density gradient dissolved in 20 mM Tris-HCL (pH 7.9),
already prepared in a 5.times.41 mm open top tube (Bekman). A
specialized adaptor was used to anchor the tube in an SW55Ti rotor
for centrifugation at 50,000 rpm, 4.degree. C. for one hour. After
centrifugation, 55 .mu.l of each solution was fractionated from the
top of the tube, and the 12th fraction was obtained by adding 20 mM
Tris-HCl (pH 7.9) to the remaining sample to 55 .mu.l and
recovering as a wash from the bottom of the tube. After separating
10 .mu.l of each fraction by SDS-PAGE, Western blotting was
performed with anti-VP1 polyclonal antibody and anti-GFP monoclonal
antibody (Roche). If VLP is formed in the insect cells, a peak for
VP1 protein is detected in the 7th to 10th fractions. Detection of
a peak for the GFP fusion protein in the 7th to 10th fractions
together with a peak for VLP suggests that it has been included in
the formed VLP. Inclusion of the fusion protein in the virus-like
structures was examined in this manner.
[0085] The results are shown in FIG. 11.
[0086] As clearly shown by these results, with fusion of GFP to the
C-terminal end of a VP2 protein fragment comprising at least the
VP1-binding region, the fusion protein could be taken up by
virus-like structures formed from VP1.
[0087] This result suggests that if a bioactive substance of
interest, instead of GFP, is linked to VP2 or a fragment thereof
containing the VP1-binding region, the bioactive substance can be
taken up into virus-like structures.
Example 6
In Vitro Incorporation of DNA into Virus-Like Structures
[0088] SV40 VP1 protein and DNA were combined in a weight ratio of
VP1:DNA=600:0-1, and after cooling for 30 minutes on ice, the
mixture was dialyzed against a solution containing 150 mM NaCl and
2 mM CaCl.sub.2 (pH 5) at room temperature for 16 hours. A portion
of the dialysate was used for electron microscope observation and
for protein quantitation (input protein), while another portion was
subjected to centrifugation using a sucrose cushion and the
particulate substance and DNA were recovered. The recovered
substance was used for measurement of (1) protein (amount of
protein forming particles), (2) the amount of DNA after
decomposition of protein with Pronase K (amount of input DNA) and
(3) the amount of DNA remaining after decomposition removal of
non-encapsulated DNA by DNase and decomposition of protein by
Pronase K (DNA encapsulated in particles).
[0089] The results are shown in FIGS. 12 and 13. As clearly seen
from FIG. 12, DNA was incorporated into VP1 virus-like structures
when the amount of DNA was at least 0.2 part by weight to 600 parts
by weight of VP1 protein.
Example 7
Formation of Virus-Like Particles Incorporating RNA
[0090] RNA was mixed with purified VP1 pentamer protein. The
mixture was solvent-exchanged at room temperature at physiological
conditions using a dialysis method. An electron microscope was used
for observation of the aggregated state of VP1 pentamers in the
exchanged solvent.
[0091] For example, 20 .mu.l of 500 ng/.mu.l concentration VP1
pentamer and 0.79 .mu.l of 938.7 ng/.mu.l concentration total RNA
were combined and adjusted to a volume of 100 .mu.l with a 20 mM
Tris-HCl (pH 7.9), 150 mM NaCl, 5 mM EGTA, 5 mM DTT solution. The
solution was incubated at 4.degree. C., 30 min and exchanged with a
pH 5, 150 mM NaCl, 2 mM CaCl.sub.2 solution by dialysis for 16
hours at room temperature. The solution was recovered, and an
electron microscope was used for observation of the aggregated
state of VP1 pentamers in the exchanged solvent. The results are
shown in FIG. 14. It is seen that addition of total RNA formed
globular virus-like particles.
INDUSTRIAL APPLICABILITY
[0092] Particles cannot be formed by allowing high-concentration
SV40 VP1 pentamers to stand under physiological conditions. For
example, when approximately 80 ng/.mu.L of VP1 pentamer is allowed
to stand in a pH 5 to 7 solution containing 150 mM NaCl, 2 mM
CaCl.sub.2, only tube-like structures or amorphous aggregates are
formed. However, addition of VP2 protein at a concentration of
approximately 15 to 20 ng/.mu.L under the same conditions can
efficiently form uniform-sized particles. Formation of
uniform-sized virus-like particles was also observed under high pH
conditions of pH 8.0 to 10.0, without VP2 protein. In other words,
it has become possible to produce viral particle-like structures
under the physiological conditions of low salt concentration and a
pH between 5 and 10.
Sequence CWU 1
1
61352PRTSimian virus 40 1Met Gly Ala Ala Leu Thr Leu Leu Gly Asp
Leu Ile Ala Thr Val Ser1 5 10 15Glu Ala Ala Ala Ala Thr Gly Phe Ser
Val Ala Glu Ile Ala Ala Gly 20 25 30Glu Ala Ala Ala Ala Ile Glu Val
Gln Leu Ala Ser Val Ala Thr Val 35 40 45Glu Gly Leu Thr Thr Ser Glu
Ala Ile Ala Ala Ile Gly Leu Thr Pro 50 55 60Gln Ala Tyr Ala Val Ile
Ser Gly Ala Pro Ala Ala Ile Ala Gly Phe65 70 75 80Ala Ala Leu Leu
Gln Thr Val Thr Gly Val Ser Ala Val Ala Gln Val 85 90 95Gly Tyr Arg
Phe Phe Ser Asp Trp Asp His Lys Val Ser Thr Val Gly 100 105 110Leu
Tyr Gln Gln Pro Gly Met Ala Val Asp Leu Tyr Arg Pro Asp Asp 115 120
125Tyr Tyr Asp Ile Leu Phe Pro Gly Val Gln Thr Phe Val His Ser Val
130 135 140Gln Tyr Leu Asp Pro Arg His Trp Gly Pro Thr Leu Phe Asn
Ala Ile145 150 155 160Ser Gln Ala Phe Trp Arg Val Ile Gln Asn Asp
Ile Pro Arg Leu Thr 165 170 175Ser Gln Glu Leu Glu Arg Arg Thr Gln
Arg Tyr Leu Arg Asp Ser Leu 180 185 190Ala Arg Phe Leu Glu Glu Thr
Thr Trp Thr Val Ile Asn Ala Pro Val 195 200 205Asn Trp Tyr Asn Ser
Leu Gln Asp Tyr Tyr Ser Thr Leu Ser Pro Ile 210 215 220Arg Pro Thr
Met Val Arg Gln Val Ala Asn Arg Glu Gly Leu Gln Ile225 230 235
240Ser Phe Gly His Thr Tyr Asp Asn Ile Asp Glu Ala Asp Ser Ile Gln
245 250 255Gln Val Thr Glu Arg Trp Glu Ala Gln Ser Gln Ser Pro Asn
Val Gln 260 265 270Ser Gly Glu Phe Ile Glu Lys Phe Glu Ala Pro Gly
Gly Ala Asn Gln 275 280 285Arg Thr Ala Pro Gln Trp Met Leu Pro Leu
Leu Leu Gly Leu Tyr Gly 290 295 300Ser Val Thr Ser Ala Leu Lys Ala
Tyr Glu Asp Gly Pro Asn Lys Lys305 310 315 320Lys Arg Lys Leu Ser
Arg Gly Ser Ser Gln Lys Thr Lys Gly Thr Ser 325 330 335Ala Ser Ala
Lys Ala Arg His Lys Arg Arg Asn Arg Ser Ser Arg Ser 340 345
3502364PRTSimian virus 40 2Met Lys Met Ala Pro Thr Lys Arg Lys Gly
Ser Cys Pro Gly Ala Ala1 5 10 15Pro Lys Lys Pro Lys Glu Pro Val Gln
Val Pro Lys Leu Val Ile Lys 20 25 30Gly Gly Ile Glu Val Leu Gly Val
Lys Thr Gly Val Asp Ser Phe Thr 35 40 45Glu Val Glu Cys Phe Leu Asn
Pro Gln Met Gly Asn Pro Asp Glu His 50 55 60Gln Lys Gly Leu Ser Lys
Ser Leu Ala Ala Glu Lys Gln Phe Thr Asp65 70 75 80Asp Ser Pro Asp
Lys Glu Gln Leu Pro Cys Tyr Ser Val Ala Arg Ile 85 90 95Pro Leu Pro
Asn Leu Asn Glu Asp Leu Thr Cys Gly Asn Ile Leu Met 100 105 110Tyr
Glu Ala Val Thr Val Lys Thr Glu Val Ile Gly Val Thr Ala Met 115 120
125Leu Asn Leu His Ser Gly Thr Gln Lys Thr His Glu Asn Gly Ala Gly
130 135 140Lys Pro Ile Gln Gly Ser Asn Phe His Phe Phe Ala Val Gly
Gly Glu145 150 155 160Pro Leu Glu Leu Gln Gly Val Leu Ala Asn Tyr
Arg Thr Lys Tyr Pro 165 170 175Ala Gln Thr Val Thr Pro Lys Asn Ala
Thr Val Asp Ser Gln Gln Met 180 185 190Asn Thr Asp His Lys Ala Val
Leu Asp Lys Asp Asn Ala Tyr Pro Val 195 200 205Glu Cys Trp Val Pro
Asp Pro Ser Lys Asn Glu Asn Thr Arg Tyr Phe 210 215 220Gly Thr Tyr
Thr Gly Gly Glu Asn Val Pro Pro Val Leu His Ile Thr225 230 235
240Asn Thr Ala Thr Thr Val Leu Leu Asp Glu Gln Gly Val Gly Pro Leu
245 250 255Cys Lys Ala Asp Ser Leu Tyr Val Ser Ala Val Asp Ile Cys
Gly Leu 260 265 270Phe Thr Asn Thr Ser Gly Thr Gln Gln Trp Lys Gly
Leu Pro Arg Tyr 275 280 285Phe Lys Ile Thr Leu Arg Lys Arg Ser Val
Lys Asn Pro Tyr Pro Ile 290 295 300Ser Phe Leu Leu Ser Asp Leu Ile
Asn Arg Arg Thr Gln Arg Val Asp305 310 315 320Gly Gln Pro Met Ile
Gly Met Ser Ser Gln Val Glu Glu Val Arg Val 325 330 335Tyr Glu Asp
Thr Glu Glu Leu Pro Gly Asp Pro Asp Met Ile Arg Tyr 340 345 350Ile
Asp Glu Phe Gly Gln Thr Thr Thr Arg Met Gln 355 36035PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Leu
Pro Leu Leu Leu1 545PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Ala Pro Leu Leu Ala1 555PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Leu
Arg Leu Leu Leu1 565PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 6Ala Arg Leu Leu Ala1 5
* * * * *